Transmission of HIV during breastfeeding remains the most significant challenge for the prevention of mother-to-child HIV transmission (MTCT) worldwide. Although short-course antiretroviral regimens can reduce transmission to as low as 2-4% during the antenatal and intrapartum periods, these gains may be lost during breastfeeding, which poses a > 9% absolute risk of transmission by 18 months. HIV-infected mothers living in developing countries often do not have the choice to formula-feed due to cost, limited access to clean water, and strong cultural traditions. In addition, formula-feeding leads to excess morbidity and mortality throughout much of the developing world. Maternal highly active antiretroviral therapy (HAART) reduces the cell-free source of virus in breast milk, but does not lower cell-associated virus. Both may be responsible for breastfeeding transmission, and their relative contributions are critical to our understanding of whether maternal HAART can reduce breastfeeding MTCT. The cell types that serve as reservoirs for HIV in breast tissue and the mammary gland's role in perpetuating the virus are largely unknown. Our preliminary data suggest that mammary secretory alveolar luminal cells play a role in transferring the virus to susceptible CD4+ cells which enter breast milk. To develop these findings further, and to delineate mechanisms by which mammary epithelial cells may contribute to a cell-associated HIV-1 viral reservoir in the breast compartment, we will test the hypotheses that (1) mammary epithelial cells (MEC) are susceptible to infection with HIV-1 and/or can initiate a productive infection in susceptible CD4+ lymphocytes and macrophages; (2) HIV-infected CD4+ T lymphocytes and macrophages facilitate infection by cell-associated trancytosis of virus across MEC and, (3) innate immune factors secreted by MEC induce recruitment, proliferation, and HIV replication in CD4+ target cells. Understanding the source(s) of cell-associated virus in breast milk may contribute to clinical decisions regarding the initiation and duration of HAART for HIV-infected pregnant and lactating women in developing countries. The applicant has designed a career development plan that includes didactic, administrative, and hands-on laboratory training, with the intent to translate these skills and experimental findings into a competitive R-level proposal. This K08 project will take place within the context of expert mentorship and strong institutional support.